Tuberculosis (TB) remains a leading cause of infectious disease morbidity and mortality. While incremental advances in sputum-based TB diagnostic tests have been made, the ability to rapidly detect M. tuberculosis (MTB) antigens with high sensitivity in clinically accessible specimens such as blood and urine would transform the TB diagnostic process. MTB lipids are attractive diagnostic targets, and while several lines of evidence indicate that certain MTB lipids are present in blood and urine in TB patients, the biology remains unexplored and the diagnostic potential unrealized. Our team has developed a waveguide-based optical biosensor platform for sensitive detection of MTB lipoarabinomannan (LAM). Further, we have developed a novel ultrasensitive detection strategy for monomeric MTB LAM in serum, based on its amphiphilic nature and consequent interaction with supported lipid bilayers. Preliminary work with a small number of clinical specimens is promising. Moreover, in vitro studies have shown that LAM in serum is packaged in lipid assemblies with human lipoproteins, especially high-density lipoprotein (HDL), raising the possibility that human lipoproteins serve as sinks for amphiphilic MTB products. By exploiting this interaction of amphiphilic LAM, we have developed a strategy for selective pull-down of LAM from serum; this novel strategy could be applied to other biomarkers. The overall goals of this R21 project are a) to refine our approach for detection of LAM in clinical specimens using existing serum and urine specimens from adults with well-characterized TB disease status, and b) to extend preliminary in vitro observations on the interaction of LAM with human lipoproteins to clarify the biodistribution of LAM and determine if other human lipoproteins are associated with LAM. The proposed studies could lead to a breakthrough not only with respect to novel ultrasensitive approaches for TB diagnosis through detection of MTB lipids, but also with respect to TB and human biology since the role of human plasma lipoproteins in mycobacterial lipid trafficking is unexplored. The proposed exploratory studies are expected to lay the groundwork for development of a rapid diagnostic testing platform as well as future comprehensive, hypothesis-based studies aimed at elucidating the biology and therapeutic implications of LAM and other mycobacterial pathogen-associated molecular patterns. The feasibility of this project is enhanced by the existing clinical specimens for study, as well as the multidisciplinary team including engineers, chemists, and TB clinicians. This project arose directly from technology-seeking activities of investigators in the NIAID/DMID-funded TB Clinical Diagnostics Research Consortium contract.